Methods for improved in vitro - in vivo efficacy determination

ABSTRACT

A method is presented for treating herpes simplex virus (HSV) infection comprising: (a) locally administering a substance that induces a delayed type hypersensitivity (DTH) response to a patient at a site of an HSV lesion to induce a DTH response at the site of the lesion during one or more outbreaks of the HSV infection.

BACKGROUND

Herpes simplex virus (HSV) causes painful lesions on the skin or mucousmembranes characterized by vesicles filled with a clear fluid. HSV type1 (HSV-1) commonly infects the mouth, face, and eyes. HSV type 2commonly infects the genitals and buttocks. But each serotype can causeinfection in all these locations. (Stalkup J R et al., Chapter 80, HumanHerpesviruses in Dermatology, Bolognia J L et al. eds. 2003, MosbyEdingurgh, United Kingdom).

Primary infection with HSV typically causes mild fever and lesions atthe site of infection. Healing occurs in 8-12 days on average, whereuponthe virus migrates to nerve ganglia, where it resides in a latent phase.The virus can be activated again by multiple causes, including physicalor emotional stress, colds, fever, immune suppression, or no apparentcause. Activation results in secondary outbreaks. For HSV-1, thisusually involves cold sores on the vermillion border of the lips. ForHSV-2, the secondary outbreak most commonly causes lesions in or aroundthe genitals, including the vulva, vagina, or penis.

Itching, tingling, and a burning sensation usually precede localizederythema of the skin or mucous membranes by a few hours. Vesicles thenform on the skin or mucous membranes. After a few days the ulcers dryand become crusted and generally heal in about 10 days.

The outbreaks cause local pain and a mild generalized fever in manycases. HSV-2 is generally transmitted by sexual contact. HSV-1 isthought to be transmitted by contact with saliva containing the virus.Infection with one or both serotypes of HSV is extremely common. Somehave estimated that 90% of the world's population have antibodies toHSV-1. Forty to sixty million persons in the U.S. are infected withHSV-2. (Stalkup J R et al., Supra.)

There is no cure for HSV infection. Antiviral therapy marginally reducesviral shedding and symptoms in secondary outbreaks. Antiviraltherapeutics can heal chronic infection in immunocompromised patients.Antiviral therapeutics are also used prophylactically. But antivirals donot cure the infection, and prophylactic antiviral therapy therefore maybe needed for a patient's entire life. (Chakrabarty A et al. 2005, SkinTherapy Lett. 10(1):1-4.) Even with prophylactic use of antivirals,outbreaks still typically occur, although at a lower frequency. Commonlyused antivirals for HSV include acyclovir and its derivatives, e.g.,valacyclovir and famciclovir. They are usually given orally, but canalso be administered by i.v. or in a topical cream. Docosanol cream(ABREVA) has also been shown to decrease the duration of outbreaksslightly (Sacks S L et al. 2001, J Am Acad Dermatol. 45(2):222-30).

New substances and methods to treat HSV infection are needed.

SUMMARY

One embodiment of the invention involves administering a substance thatinduces a delayed-type hypersensitivity (DTH) reaction locally at a siteof an HSV lesion during an outbreak. This causes T cells to swarm thearea of the lesion, which has a high concentration of virus. Thisappears to train the immune system to recognize the herpes simplex virusand strengthens the immune response to the virus, which lessens theseverity and frequency of subsequent HSV outbreaks (e.g., cold sores orgenital outbreaks) after the treated outbreak or outbreaks. Any compoundor mixture that induces a DTH response can be used, including squaricacid dibutyl ester (SADBE), diphenylcyclopropenone (DPCP),1-chloro-2,4-dinitrobenzene (DNCB), and extract of poison ivy leaves.Protein antigens that induce a DTH response, such as mumps antigen orother antigens a patient might have immunity against, can also beinjected into the skin or mucous membrane at the site of an outbreak.The invention has been tested with cold sores, which are caused byherpes simplex virus type 1. It also works with genital herpes, causedby herpes simplex virus type 2.

One embodiment of the invention involves a method of treating herpessimplex virus (HSV) infection comprising: locally administering asubstance that induces a delayed type hypersensitivity (DTH) response toa patient at a site of an HSV lesion to induce a DTH response at thesite of the lesion during one or more outbreaks of the HSV infection.

Another embodiment of the invention provides a medical use of asubstance capable of inducing a delayed-type hypersensitivity responsein humans to prepare a medicament effective to reduce the frequency orseverity of subsequent HSV outbreaks in a patient with HSV infection.

It has now very surprisingly been found in a clinical trial (describedin Example 2 below) with persons that suffer frequent cold sores, herpeslabialis lesions on the lip, that topically applying on the inner aspectof the upper arm a substance that induces a DTH response, squaric aciddibutyl ester (SADBE) actually prevented future HSV outbreaks. Patientshad topical application of SADBE just once on the upper arm withoutfurther application at the site of an HSV lesion, and this actuallyprevented future outbreaks. The result was statistically significantversus a placebo control group.

This means we can prevent future HSV outbreaks by administering asubstance that induces a DTH response at a site other than the site ofan HSV outbreak.

The substances that can induce a DTH response are generally in twoclasses: (1) topical immunosensitizers, such as SADBE, that when appliedtopically, for instance in a solution in DMSO, induce a DTH response;and (2) recall antigens, such as Candida extract or mumps antigen, thatwhen injected intradermally induce a DTH response.

Thus, one other embodiment provides a method of treating herpes simplevirus (HSV) infection comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin of a person infectedwith HSV at a site other than the genitals or lips, or to the genitalsor the lips but at a time when the person is not having an HSV outbreakon the genitals or lips; or (b) intradermally injecting a recall antigeninto a person infected with HSV at a site other than the genitals orlips, or into the genitals or the lips but at a time when the person isnot having an outbreak on the genitals or lips; wherein the method doesnot further comprise (i) applying a topical immunosensitizer to the skinof a person infected with HSV on an HSV lesion at a time of an HSVoutbreak in the person; or (ii) intradermally injecting a recall antigeninto a person infected with HSV in an HSV lesion at the time of an HSVoutbreak in the person.

Another embodiment provides a method of treating herpes simple virus(HSV) infection comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin of a person infectedwith HSV at a time when the person is not having an HSV outbreak, orapplying a topical immunosensitizer to the skin of a person infectedwith HSV during an HSV outbreak wherein the applying is at a site otherthan a site of an HSV lesion; or (b) intradermally injecting a recallantigen into a person infected with HSV at a time when the person is nothaving an HSV outbreak, or intradermally injecting a recall antigen intoa person infected with HSV at a site other than a site of an HSV lesionduring an HSV outbreak; wherein the method does not further comprise (i)applying a topical immunosensitizer to the skin of a person infectedwith HSV on an HSV lesion at a time of an HSV outbreak in the person; or(ii) intradermally injecting a recall antigen into a person infectedwith HSV in an HSV lesion at the time of an HSV outbreak in the person.

Another embodiment provides a method of treating HSV infectioncomprising: a first administering step comprising: (a) applying atopical immunosensitizer to the skin of a person infected with HSV at asite other than the genitals or lips, or to the genitals or the lips butat a time when the person is not having an HSV outbreak on the genitalsor lips; or (b) intradermally injecting a recall antigen into a personinfected with HSV at a site other than the genitals or lips, or into thegenitals or the lips but at a time when the person is not having an HSVoutbreak on the genitals or lips; and a second administering stepcomprising: repeating step (a) or step (b) by applying the same topicalimmunosensitizer or intradermally injecting the same recall antigen onthe person at least one other time at a time between 1 week and 2 yearsafter the first administering step.

Another embodiment provides a method of treating herpes simple virus(HSV) infection comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin on the inner aspect ofthe upper arm of a person infected with HSV; or (b) intradermallyinjecting a recall antigen into the skin on the inner aspect of theupper arm of a person infected with HVS; wherein the method reduces thefrequency, severity, or duration of HSV outbreaks in the person afterthe administering step.

Another embodiment provides a medical use of a substance capable ofinducing a delayed-type hypersensitivity (DTH) response in humans (atopical immunosensitizer or a recall antigen) to prepare a medicamenteffective to reduce the frequency of subsequent herpes simplex virus(HSV) outbreaks in a patient with HSV infection when administered to theskin of a person infected with HSV at a time when the person is nothaving an HSV outbreak or administered to the skin of a person infectedwith HSV during an HSV outbreak wherein the administering is at a siteother than a site of an HSV lesion.

Another embodiment provides a medical use of a topical contactsensitizer or a recall antigen to prepare a medicament effective toreduce the frequency of subsequent herpes simplex virus (HSV) outbreaksin a patient with HSV infection when administered by (a) applying atopical immunosensitizer to the skin on the inner aspect of the upperarm of a person infected with HSV; or (b) intradermally injecting arecall antigen into the skin on the inner aspect of the upper arm of aperson infected with HSV.

Another embodiment provides a method of treating HSV infectioncomprising a first administering step comprising: (a) applying a topicalimmunosensitizer to the skin of a person infected with HSV at a siteother than the genitals or lips, or to the genitals or the lips but at atime when the person is not having an outbreak on the genitals or lips;and a second administering step comprising (b) applying the same topicalimmunosensitizer onto an epithelial HSV lesion at the site of the lesionduring an HSV outbreak in the person; wherein the second administeringstep occurs at least 1 week after the first administering step; whereinthe second administering step does not produce localized erythema at thesite of administration on the person.

Another embodiment provides a composition comprising a topicalimmunosensitizer or a recall antigen for use in a method of treating HSVinfection, the method comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin of a person infectedwith HSV at a time when the person is not having an HSV outbreak, orapplying a topical immunosensitizer to the skin of a person infectedwith HSV during an HSV outbreak wherein the applying is at a site otherthan a site of an HSV lesion; or (b) intradermally injecting a recallantigen into a person infected with HSV at a time when the person is nothaving an HSV outbreak, or intradermally injecting a recall antigen intoa person infected with HSV at a site other than a site of an HSV lesionduring an HSV outbreak; wherein the method does not further comprise (i)applying a topical immunosensitizer to the skin of a person infectedwith HSV on an HSV lesion at a time of an HSV outbreak in the person; or(ii) intradermally injecting a recall antigen into a person infectedwith HSV in an HSV lesion at the time of an HSV outbreak in the person;wherein the method reduces the frequency of new HSV outbreaks in theperson after the administering step.

Another embodiment provides a composition comprising a topicalimmunosensitizer or a recall antigen for use in a method of treating HSVinfection, the method comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin on the inner aspect ofthe upper arm of a person infected with HSV; or (b) intradermallyinjecting a recall antigen into the skin on the inner aspect of theupper arm of a person infected with HSV; wherein the method reduces thefrequency of new HSV outbreaks in the person after the administeringstep.

The methods reduce the frequency, duration, or severity outbreaks in theperson after the administering step or steps. The term “applying atopical immunosensitizer to the skin” includes intradermally injectingthe topical immunosensitizer, although usually the topicalimmunosensitizer would be applied topically.

DETAILED DESCRIPTION

One embodiment of the invention involves a method of treating herpessimplex virus (HSV) infection comprising: locally administering asubstance that induces a delayed type hypersensitivity (DTH) response toa patient at a site of an HSV lesion to induce a DTH response at thesite of the lesion during one or more outbreaks of the HSV infection. An“outbreak” refers to a temporal episode of burning, tingling, painful,or visible lesions arising from HSV infection. Outbreaks are separatedby dormant periods when the patient has no symptoms of infection.

A delayed-type hypersensitivity (DTH) response, also known as a type IVhypersensitivity response, is an immune response that typically reachesmaximal intensity 24-48 hours after contact with an immunogen. Itproduces visible erythema at the site of contact with the immunogen. ADTH response is primarily a T cell response (Goldsby, Richard A. et al.2000, Kuby Immunology, 4th edition, WH Freeman and Co., New York,chapter 16). It occurs when antigen activates sensitized T_(DTH) cells.Activation of the T_(DTH) cells results in secretion of severalcytokines, which draws macrophages into the area and activates them.

T cells and cell-mediated immunity are the primary arm of the immunesystem responsible for fighting viral infection. Cell-mediated immunityis responsible for recognizing and eliminating cells that harborintracellular pathogens, such as HSV. Without wishing to be bound bytheory, the inventors believe the DTH response of the present inventioncauses T cells to swarm the area of an HSV outbreak. With a large numberof HSV viral particles and HSV proteins in the area during an outbreak,the T cells better learn to recognize HSV and cells harboring HSV,resulting in a stronger immune response to HSV in the future that does abetter job of ridding the body of the virus and viral-infected cells,thus preventing outbreaks and lessening the severity of outbreaks. Thisis a process of “epitope unveiling” whereby epitopes that were poorlyrecognized by the immune system become better recognized.

It has previously been shown that warts caused by human papilloma viruscan be treated by inducing a DTH response at the site of a wart withtopical application of contact sensitizers, such as DNCB, or withintralesional injection of protein antigens unrelated to human papillomavirus that induce a DTH response in the patient, such as mumps antigen,candida antigen, or trichophyton antigen. (U.S. Pat. No. 6,350,451; U.S.published patent application no. 20050175634; Johansson, E. et al. 1984,Dinitrochlorobenzene (DNCB) treatment of viral warts, Acta Derm.Verereol (Stockh) 64:529-533; Dunnigan, W. G. et al., 1982,Dinitrochlorobenzene immunotherapy for verrucae resistant to standardtreatment modalities, J. Am. Acad. Dermatol. 6:40-45.) The presentinvention appears to work by a similar mechanism.

The immunogen of the present invention used to induce the DTH responsecan be a protein antigen that the subject has previously encountered andto which he or she has developed immune recognition. This is theprinciple of skin tests for allergens or antigens such as thetuberculosis antigen. The immunogen can also be a topical contactsensitizer, such as urushiol, an oil that is the active irritatingingredient in poison ivy, poison oak, and other irritating plants.Topical contact sensitizers are typically haptens. Haptens are smallmolecules that do not induce an immune response on their own, but caninduce an immune response and antibodies that specifically recognize thehapten determinant when attached to proteins or other macromolecules.Topical contact sensitizers are typically haptens that react withproteins in the skin to form adducts that are immunogenic.

Thus, in some embodiments the substance that induces a DTH response is atopical contact sensitizer—a substance that when applied topically tohuman skin induces a DTH response. Topical contact sensitizers are alsoreferred to herein as topical immunosensitizers. The two terms have thesame meaning and are used interchcangeably.

Thus, in some embodiments the method involves topically applying asubstance that induces a DTH response to a patient at a site of an HSVlesion to induce a DTH response at the site of the lesion.

A topically applied contact sensitizer is typically applied as asolution in an organic solvent, e.g., acetone or dimethylsulfoxide. Ifsoluble in water, it can instead by applied in an aqueous solution. Itcan also be applied in a cream, ointment, lotion, oil, etc.

In specific embodiments where the substance that induces a DTH responseis applied topically (is a topical contact sensitizer) the substance issquaric acid dibutyl ester (SADBE), diphenylcyclopropenone (DPCP),1-chloro-2,4-dinitrobenzene (DNCB), or 1-chloro-2,6-dinitrobenzene. Insome embodiments, the substance is squaric acid or an ester thereof. Ina more specific embodiment, it is a squaric acid diester, for instance,squaric acid diethyl ester or squaric acid dibutyl ester (SADBE).

The structure of squaric acid is shown below.

SADBE has the systemic name of 3,4-Dibutoxy-3-cyclobutene-1,2-dione. Itsstructure is shown below:

These contact sensitizers are available from several commercial sources,including Spectrum Chemicals & Laboratory Products, a division ofSpectrum Chemical Manufacturing Corp., Gardena, Calif. and NewBrunswick, N.J.

In other embodiments, the substance that induces a DTH responsecomprises urushiol or an extract of an irritating plant.

The term “topical contact sensitizer” as used herein has the samemeaning as “topical immunosensitizer.” The two terms mean any substancethat when topically applied to human skin induces a localized DTHresponse (as evidenced by local erythema at the site of administrationthat peaks in a delayed manner, for instance, at about 2 days afteradministration, rather than peaking within the first 24 hours ofadministration) in a majority of previously sensitized persons. Anappropriate test is application of a test substance at a concentrationof 2% w/v in acetone or DMSO in a volume of 0.2 ml to a skin area of 1cm squared or less (test application), after prior application of thesame substance in the same concentration and amount to the same person(sensitization application) in the period of 2-12 weeks prior to thetest application. For a topical contact sensitizer, the test applicationwill induce a localized DTH response in the majority of persons.

In other embodiments, the substance that induces a DTH response is aprotein antigen. The protein antigen is typically not an HSV antigen,although it could be. Any antigen that induces a DTH response can beused. A subject may have a preexisting sensitivity to the antigen. Mumpsantigen, candida antigen, and trichophyton antigen are three preferredantigens in this regard, since a large percentage of the population hasa preexisting sensitivity to one or more of these antigens. Otherforeign (non-self) antigen to which a subject does not have apreexisting sensitivity can also be used. In that case, the subjectshould be sensitized to the antigen by administering the antigen to thesubject (i.e., immunizing the subject with the antigen) before locallyadministering the antigen to the subject at a site of HSV outbreak toinduce a DTH response at the site of an outbreak.

In specific embodiments, the substance that induces a DTH response is arecall antigen.

The prototypical recall antigens are those commonly used in immunologicskin testing to test immune response, particularly mumps antigen,candida antigen, and trichophyton antigen. The test shows if the body“remembers” or “recalls” the antigen, i.e., has a delayed-typehypersensitivity response in the skin where the antigen was administeredby intradermal injection.

The term “recall antigen” is defined herein as a substance or mixturecontaining a plurality of proteinaceous antigens, wherein the substanceor mixture induces a delayed-type hypersensitivity response (asevidenced by delayed appearance of erythema) in intradermal skin test ina majority of immunocompetent people previously sensitized or exposed tothe recall antigen. The prototypical recall antigens are those commonlyused in immunologic skin testing to test immune response, particularlymumps antigen, candida antigen, and trichophyton antigen. Each of these,although referred to by the singular term “antigen” is actually composedof several or many molecular substances that can induce an immuneresponse.

In specific embodiments, the recall antigen may be mumps antigen (e.g.,killed whole mumps virus), Candida extract, or Trichophyton extract.

In specific embodiments, the recall antigen is killed whole virus,killed whole bacteria, or killed whole microorganisms.

Thus, in some embodiments, the method involves intralesionally (e.g.,intradermally) injecting the substance that induces a DTH response intoa patient at a site of an HSV lesion in the patient to induce a DTHresponse at the site of the lesion.

In a specific embodiment of intralesionally injecting the substance, thesubstance comprises mumps antigen, candida antigen, or trichophytonantigen.

The dosages of antigen can be approximately the same as the dosages usedin skin tests with these antigens. The dose should be a dose thatinduces a mild to moderate DTH response.

In a specific embodiment, the method further comprises before inducing aDTH response at the site of an outbreak, administering the substancethat induces a DTH response to the patient to develop a sensitivity tothe substance in the patient. This presensitization step can be donewith intralesionally injected antigens or with topically appliedsubstances. For the presensitization step, the substance can beadministered at the site of outbreak. This may be beneficial to helpstimulate recognition of HSV and HSV-infected cells. Or for thepresensitization step, the substance can be administered anywhere. Forinstance, a topical contact sensitizer may be administered on theforearm, or an injectable antigen may be administered by intradermalinjection to the forearm, during a time when the patient may or may notbe suffering an outbreak.

In a specific embodiment, the method can involve, before the step ofinducing a DTH response at the site of an HSV lesion, administering thesubstance that induces a DTH response to the patient at at least onedosage level to determine a dosage level of the substance to administerto induce a DTH response at the site of an HSV lesion.

The lips and genitals, which are the most common areas for lesions ofHSV-1 and HSV-2 outbreaks respectively, are both very sensitive areas.The lips are also a very visible area. Thus, it is desirable to notinduce a severe DTH response in these sites because such a severeresponse can be painful and unsightly. Therefore, one may want to testone or more dosages of the substance in a less sensitive and lessvisible area, such as the forearm, to find a dose that induces anappropriate level of DTH response before applying the substance at thesite of an HSV lesion. It has been found that the method works even whenthe DTH response at the site of an HSV lesion is fairly mild. It doesnot appear to be necessary to induce a severe DTH response. But themethods may be more effective in inducing a sustained immunity to HSVthat prevents subsequent outbreaks if the DTH response is stronger.

It is not essential to induce a DTH response for the methods to work. Inour clinical trial described in Example 2, the treated patients who didnot develop a visible erythema at the site of application of SADBEnonetheless had a longer median time to the next HSV outbreak thanpatients receiving placebo.

Thus, in all methods described herein where the method involves inducinga DTH response, it can involve administering a substance (topicalimmunosensitizer or recall antigen) capable of inducing a DTH response,without actually inducing a visible DTH response. This is true whetherthe administering is at the site of an HSV lesion or elsewhere on theperson's body.

For DNCB, a suitable sensitizing dose appears to be a 2% DNCB solutionin acetone, dimethylsulfoxide (DMSO), or another solvent, and a suitabletreatment dose is, for example, 0.05 to 2.0% DNCB. A suitablesensitizing dose of SADBE is a 2% solution, and a suitable treatmentdose is, for example, a 0.05%-2% solution. Doses that are higher orlower than these ranges can also be used in some patients.

The topical contact sensitizer solution in one embodiment is applied bydabbing a cotton-tipped swab that has been saturated with the solutiononto the skin or mucous membrane at the desired site of application,without repeated rubbing or spreading the solution over an extendedarea. For both the sensitization and treatment applications, the topicalcontact sensitizer is preferably left on the skin for at least a fewhours before washing it off.

In another embodiment, the contact sensitizer solution is applied with afixed volume device, such as a micropipette, syringe, or microsyringe.This allows application of a defined volume and therefore a definedamount of the contact sensitizer. That can be helpful to produce a morepredictable level of intensity of the DTH response. A typical volume is2-40 microliters to be able to apply the solution to a targeted andlimited area such as a lesion without the solution spreading or runningto nontarget areas.

In particular embodiments of the methods, the herpes simplex virus isHSV type 1. In other embodiments, it is HSV type 2.

In particular embodiments, the method involves administering thesubstance that induces a DTH response at the site of an HSV lesion on oradjacent to the lip of the patient.

In particular embodiments, the method involves administering thesubstance that induces a DTH response at the site of an HSV lesion onthe genitals of the patient.

In particular embodiments, the method involves topically applying thesubstance that induces a DTH response to skin at a site of an HSVlesion. In other embodiments, it involves topically applying thesubstance to a mucous membrane at a site of an HSV lesion.

The method can reduce the severity of future outbreaks or the frequencyof future outbreaks, or both. The method is not intended to reduce theseverity of the outbreak during which the DTH response is elicited atthe site of an HSV lesion. In fact, it may actually worsen thatparticular outbreak. The DTH response involves inflammation and canproduce a mild fever. Inflammation and fever are themselves triggers forHSV outbreaks, so the DTH response may trigger the HSV to worsen itscurrent outbreak. But once that outbreak subsides or is brought undercontrol, subsequent outbreaks are found to be much less frequent and/orsevere.

To counteract the tendency of the DTH response to worsen the HSVoutbreak during which the DTH response is induced, in one embodiment,the method can involve treating the patient with antiviral medications,such as acyclovir or valacyclovir, during the outbreak in which the DTHresponse is induced.

The step of inducing a DTH response at a site of an HSV lesion can berepeated more than once to further strengthen the immune response toHSV, if further outbreaks occur. Preferably, treatments are spaced apartby at least two weeks to allow full development of the immune responsefrom the previous treatment.

Many patients may require more than one treatment to develop a level ofimmunity that decreases the number or severity of subsequent outbreaks(i.e., outbreaks subsequent to the treatment, outbreaks in which nosubstance is administered to induce a DTH response). Preferably, theimmunity developed completely prevents subsequent outbreaks. Thus, thesubstance that induces a DTH response is administered to a lesion in oneoutbreak. When the outbreak subsides and a subsequent recurrent outbreakoccurs, the substance may be administered to a lesion again. Afteradministration of the substance during 1 to 3 or more outbreaks, thepatient may have no outbreaks for an extended period of time. But theimmunity may eventually wear off, and outbreaks may recur. A substancethat induces the DTH response can then be applied again during one ormore outbreaks as needed to develop immunity again that preventsoutbreaks.

Thus, in one embodiment the method comprises: (a) locally administeringa substance that induces a delayed type hypersensitivity (DTH) responseto a patient at a site of an HSV lesion to induce a DTH response at thesite of the lesion during one outbreak of the HSV infection (forexample, during only one outbreak over a 6-month period). In oneembodiment the method comprises: locally administering a substance thatinduces a delayed type hypersensitivity (DTH) response to a patient at asite of an HSV lesion to induce a DTH response at the site of the lesionduring one outbreak of the HSV infection; wherein the method does notcomprise administering a substance that induces a DTH response to thepatient at a site of an HSV lesion to induce a DTH response at the siteof the lesion during another outbreak within 3 months before or afterthe one outbreak.

In other embodiments, the method comprises: (a) locally administering asubstance that induces a delayed type hypersensitivity (DTH) response toa patient at a site of an HSV lesion to induce a DTH response at thesite of the lesion during two or more outbreaks of the HSV infection(for example, during two or more outbreaks in a 6-month period).

One embodiment of the invention provides a medical use of a substancecapable of inducing a delayed-type hypersensitivity response in humansto prepare a medicament effective to reduce the frequency or severity ofherpes simplex virus (HSV) outbreaks.

In particular embodiments, the substance is a topical contact sensitizercapable of inducing a DTH response in humans when administeredtopically.

In other embodiments, the substance is an antigen capable of inducing aDTH response in humans when injected intradermally.

By reducing the duration and severity of subsequent outbreaks, themethods of the invention also reduce transmission of infection. This istrue for both HSV-1 infection and HSV-2 infection, and of both genitalherpes and oral herpes.

In one embodiment of the methods, the method reduces transmission ofinfection of HSV-1.

In one embodiment of the methods, the method reduces transmission ofinfection of HSV-2.

In one embodiment of the methods of the invention, the method reducestransmission of oral herpes infection.

In one embodiment of the methods of the invention, the method reducestransmission of genital herpes infection.

In specific embodiments, the methods decrease frequency of subsequentoutbreaks by at least 50% (i.e., increase time to next outbreak aftertreatment by at least 100%). In other embodiments, the methods decreasefrequency of subsequent outbreaks by at least 75%. In other embodiments,the methods decrease frequency of subsequent outbreaks by at least 70%,at least 80%, or at least 90%. In one embodiment, the methods increasetime between outbreaks. In specific embodiments, the methods increasetime between outbreaks (e.g., increase the time from resolution of theoutbreak treated to the next subsequent outbreak, as compared to theaverage time between outbreaks previously) at least 2-fold, at least3-fold, at least 4-fold, or at least 5-fold.

The immune effects of the steps of administering the topicalimmunosensitizer or recall antigen are believed to take 2 weeks or moreto develop. And inflammation that is characteristic of the DTH responsecan take up to 3 weeks to fully dissipate. Inflammation is a knowntrigger for HSV outbreaks, so the reduction in HSV outbreaks after theadministering step or steps may not fully take effect until 3 weeksafter the administering step. So in specific embodiments, the methodsdecrease frequency of outbreaks that begin at least 21 days after theadministration step. In specific embodiments, the methods increasemedian time from day 21 following the administering step to the nextoutbreak that begins at least 21 days after the administration step byat least 2-fold, or by at least 3-fold, or by at least 4-fold.

In specific embodiments, the methods of the invention reduce theduration of subsequent outbreaks. In particular embodiments, they reducethe time to healing of lesions in subsequent outbreaks by at least 10%,at least 20%, at least 30%, or at least 40%.

The methods are believed to stimulate cell-mediated immunity.Cell-mediated immunity may be assayed by peripheral blood mononuclearcell proliferation assays, as described in Example 2 below. In specificembodiments of the methods, the method increases stimulation index ofperipheral blood mononuclear cells (as illustrated in Example 2) in aproliferation assay of stimulation by HSV particles. In specificembodiments, it increases the stimulation index from less than 30 beforetreatment to more than 30, more than 50, or more than 60 aftertreatment. In other embodiments, it increases the stimulation index fromless than 50 before treatment to more than 50 or more than 60 aftertreatment. In other embodiments, it increases the stimulation index byat least 10, at least 20, at least 30, at least 40, or at least 50. Inother embodiments, it at least doubles, at least triples, or at leastquadruples the stimulation index.

Immune response enhancers can also be used with the substance thatinduces a DTH response to enhance development of immunity against HSVwith the DTH response. One type of enhancer that can be used is acytokine. Among the cytokines that may be used are an interferon (e.g.,interferon alpha), granulocyte monocyte colony stimulating factor(GM-CSF), interleukin-2, and interleukin-12. Each of these has beenshown to promote cell-mediated immune reactions or antiviral immunereactions (Kiline M O et al. 2006, J. Immunol. 177:6962-73; Arora A. etal. 2006, J. Surg. Oncol. 94:403-412; Horn et al., US Published PatentAppl. No. 20050175634).

U.S. Published Patent Appl. 20050175634 reports intralesional injectionof unrelated antigens to induce a DTH response in warts. It reports astudy where a portion of the patients received intralesional injectionof antigens only, and others received as well intralesional injection ofinterferon alpha or GM-CSF. A larger fraction of patients receivinginterferon or GM-CSF together with the antigens responded to treatmentthan patients receiving antigen alone, although the number of subjectstreated was not enough for the differences to be statisticallysignificant.

Appropriate doses of the cytokines are known in the art or can bedetermined by experimentation to identify a dosage range that gives bestresults. A suitable dose of interferon alpha, for example, isapproximately 1 million IU administered locally.

Administration of the cytokines may be by intradermal injection at thesite of the lesion. It may also be by topical administration, e.g., inan ointment, cream, or lotion (Syed T A et al. 1995. J. Mol. Medicine73:141-144).

In another embodiment, the immune response enhancer is a pharmaceuticalagent that stimulates synthesis of cytokines. In one embodiment, it is asynthetic (i.e., not a naturally occurring molecule) pharmaceuticalagent that stimulates synthesis of cytokines. Specific examples areimiquimod and resiquimod. (Spruance S L et al. 2001, J. Infect. Dis.184:196-200; Bernstein D I et al. 2005, Clinical Infectious Disease41:808-814.)

The immune response enhancer can be administered at any suitable timethat will result in it having an effect during the DTH response. Thismay be at the same time as the time that the substance that elicits aDTH response is administered or somewhat before or after. It should beadministered before or during the DTH response.

With the discovery that application of a topical immunosensitizer to thearm of a patient at a time the patient may or may not be having an HSVoutbreak reduces the frequency of future outbreaks, even without everadministering a topical immunosenzitizer or recall antigen at the siteof an HSV lesion, another embodiment provides a method of treatingherpes simple virus (HSV) infection comprising: an administering stepcomprising: (a) applying a topical immunosensitizer to the skin of aperson infected with HSV at a time when the person is not having an HSVoutbreak, or applying a topical immunosensitizer to the skin of a personinfected with HSV during an HSV outbreak wherein the applying is at asite other than a site of an HSV lesion; or (b) intradermally injectinga recall antigen into a person infected with HSV at a time when theperson is not having an HSV outbreak, or intradermally injecting arecall antigen into a person infected with HSV at a site other than asite of an HSV lesion during an HSV outbreak; wherein the method doesnot further comprise (i) applying a topical immunosensitizer to the skinof a person infected with HSV on an HSV lesion at a time of an HSVoutbreak in the person; or (ii) intradermally injecting a recall antigeninto a person infected with HSV in an HSV lesion at the time of an HSVoutbreak in the person.

Another embodiment provides a method of treating HSV infectioncomprising: a first administering step comprising: (a) applying atopical immunosensitizer to the skin of a person infected with HSV at asite other than the genitals or lips, or to the genitals or the lips butat a time when the person is not having an HSV outbreak on the genitalsor lips; or (b) intradermally injecting a recall antigen into a personinfected with HSV at a site other than the genitals or lips, or into thegenitals or the lips but at a time when the person is not having an HSVoutbreak on the genitals or lips; and a second administering stepcomprising: repeating step (a) or step (b) by applying the same topicalimmunosensitizer or intradermally injecting the same recall antigen onthe person at least one other time at a time between 1 week and 2 yearsafter the first administering step.

Another embodiment provides a method of treating herpes simple virus(HSV) infection comprising: an administering step comprising: (a)applying a topical immunosensitizer to the skin on the inner aspect ofthe upper arm of a person infected with HSV; or (b) intradermallyinjecting a recall antigen into the skin on the inner aspect of theupper arm of a person infected with HVS; wherein the method reduces thefrequency, severity, or duration of HSV outbreaks in the person afterthe administering step.

The inner aspect of the upper arm refers to the surface of the upper armthat is in contact with the chest when a person holds their arms relaxedat their sides. This is where the initial dose of topicalimmunosensitizer was applied in the clinical trial described in Example2, where efficacy was proven. The inner aspect of the upper arm is afavorable spot for application of the substance because the applicationmay cause a rash, and the inner aspect of the upper arm is hidden byclothing, so any rash is not unsightly or embarrassing. It also is neara major lymph node in the armpit, and that may be part of the reason forthe efficacy. So it may be the inner aspect of the upper arm is aparticularly favorable spot to apply the drug for efficacy purposes, aswell as being a hidden spot on the body in case a rash develops.

Again, in specific embodiments of the methods of treating HSV describedherein, the method reduces the frequency of new HSV outbreaks in theperson after the administering step.

In specific embodiments, the method comprises applying a topicalimmunosensitizer to the skin of a person infected with HSV at a timewhen the person is not having an HSV outbreak, or applying a topicalimmunosensitizer to the skin of a person infected with HSV during an HSVoutbreak wherein the applying is at a site other than a site of an HSVlesion.

In specific embodiments, the administering step comprises applying thetopical immunosensitizer to skin on the inner aspect of the upper arm ofthe person or intradermally injecting a recall antigen into skin on theinner aspect of the upper arm of the person.

In specific embodiments, the topical immunosensitizer comprises squaricacid, a squaric acid ester, diphenylcyclopropenone (DPCP),1-chloro-2,4-dinitrobenzene (DNCB), or 1-chloro-2,6-dinitrobenzene.

In specific embodiments, the topical immunosensitizer is SADBE and isapplied as a solution in DMSO.

In specific embodiments, the treated person has herpes labialis.

In specific embodiments, the person has genital herpes.

In specific embodiments, the method decreases time to healing of lesionsin a subsequent outbreak of the HSV infection after step (a) by at least30%.

In specific embodiments, the method increases time between outbreaks byat least 2-fold.

In specific embodiments, the method increases time between outbreaks byat least 4-fold.

In specific embodiments, the method at least doubles stimulation indexof peripheral blood mononuclear cells (PBMC) from a patient in aproliferation test with stimulation of proliferation by killed HSVparticles as compared to the stimulation index of PBMC from the patientbefore treatment.

In specific embodiments, the method increases median time to the nextoutbreak after the administering step by at least 2-fold, at least3-fold, or at least 4-fold.

In specific embodiments, the method increases median time from day 21following the the administering step to the next outbreak that begins atleast 21 days after the administering step by at least 2-fold, at least3-fold, or at least 4-fold.

Again, one embodiment provides a method of treating herpes simple virus(HSV) infection comprising: a first administering step comprising: (a)applying a topical immunosensitizer to the skin of a person infectedwith HSV at a time when the person is not having an HSV outbreak, orapplying a topical immunosensitizer to the skin of a person infectedwith HSV during an HSV outbreak wherein the applying is at a site otherthan a site of an HSV lesion; or (b) intradermally injecting a recallantigen into a person infected with HSV at a time when the person is nothaving an HSV outbreak, or intradermally injecting a recall antigen intoa person infected with HSV at a site other than a site of an HSV lesionduring an HSV outbreak; wherein the method does not further comprise (i)applying a topical immunosensitizer to the skin of a person infectedwith HSV on an HSV lesion at a time of an HSV outbreak in the person; or(ii) intradermally injecting a recall antigen into a person infectedwith HSV in an HSV lesion at the time of an HSV outbreak in the person.

In specific further embodiments of this, the method further comprises asecond administering step comprising: (a) applying a topicalimmunosensitizer to the skin of a person infected with HSV at a timewhen the person is not having an HSV outbreak, or applying a topicalimmunosensitizer to the skin of a person infected with HSV during an HSVoutbreak wherein the applying is at a site other than a site of an HSVlesion; or (b) intradermally injecting a recall antigen into a personinfected with HSV at a time when the person is not having an HSVoutbreak, or intradermally injecting a recall antigen into a personinfected with HSV at a site other than a site of an HSV lesion during anHSV outbreak.

In specific embodiments, the second administering step occurs 7 days to30 days after the first administering step.

In specific embodiments, the second administering step occurs 7 days to365 days after the first administering step and occurs at a time whenthe person is having an HSV outbreak (but involves administering at asite other than a site of an HSV lesion during the HSV outbreak).

In specific embodiments, the first and second administering steps bothcomprise: (a) applying a topical immunosensitizer to the skin of aperson infected with HSV at a time when the person is not having an HSVoutbreak, or applying a topical immunosensitizer to the skin of a personinfected with HSV during an HSV outbreak wherein the applying is at asite other than a site of an HSV lesion.

In specific embodiments, the first or subsequent administering steps inproduce an erythema in the person at the site of administering. In otherembodiments, the first or subsequent administering steps do not producean erythema in the person at the site of administration.

In specific embodiments where the topical immunosensitizer or recallantigen are administered at a site other than the site of an HSV lesion,the administering step comprises applying the topical immunosensitizerto skin on the spine of the person or skin over the trigeminal ganglionof the person or intradermally injecting a recall antigen into skin onthe spine of the person or skin over the trigeminal ganglion of theperson. HSV is known to reside in the spine and in the trigeminalganglion, so these may be effective sites for administering the topicalimmunosensitizer or the recall antigen.

The invention will now be illustrated by the following examples. Theexamples are intended to illustrate the invention but not limit itsscope.

EXAMPLES Example 1 Treatment of an Individual Suffering from Frequentand Severe Cold Sores

The individual treated was one of the inventors. He is a male and was 42years old at the time of this treatment. The subject suffered fromfrequent cold sores on the lower lip. The outbreaks typically lasted7-10 days and frequently longer. The subject had suffered from frequentcold sores most of his life. During the 6 months prior to thistreatment, the outbreaks had been almost continuous. After getting overone outbreak, the next outbreak would begin often within a week.

The subject was presensitized to DNCB by dipping a cotton-tipped swab ina 2% DNCB solution in acetone, and contacting the cotton-tipped swabwith two spots on the forearm. No rash developed. Two weeks later, thesubject again applied a 2% DNCB solution to his forearm at two smallspots different from where the sensitizing dose had been applied. A verystrong but localized rash ensued and lasted for 3 weeks. The subjectthen applied a 0.1%, 0.2%, and 0.3% solutions to separate small spots onthe forearm. The 0.1% solution produced almost no erythema. The 0.2%solution produced a mild but easily seen erythema that began 2 daysafter applying the solution and lasted about 3 days. Based on thisresult, the 0.2% solution was chosen as the dosage to apply to the nextcold sore.

Upon the next HSV outbreak that produced a cold sore on the subject'slower lip, the subject applied a 0.2% DNCB solution in acetone to thesite of the cold sore on his lower lip and left it on overnight. In themorning he washed it off. A mild erythema in the area of the cold soredeveloped over 2-3 days and lasted a further 5 days, and the cold soreresolved in about 7 days, which was a typical duration for thisindividual or a slightly shorter duration than normal. Following thistreatment, the subject experienced outbreaks of cold sores on his lowerlip approximately weekly for the next 4 weeks, but they were much lesssevere and of much shorter duration than before. Each outbreak wouldbegin and completely resolve in one day. Approximately two months afterthe first application of DNCB to his lip, the subject had a mild coldsore and applied the 0.2% DNCB solution in acetone to the site of an HSVlesion on his lower lip as soon as the outbreak began. This time, theinflammation and erythema with the application were more severe thanthey had been with the first application of DNCB to the lesion on hislip two months previously. We think this may be because the immunesystem was recognizing not just the DNCB but also the herpes virusantigens with this second application. With the greater inflammation,the cold sore outbreak was worse than any of the outbreaks between thefirst and second DNCB treatments. Those outbreaks had been very mild,but this HSV outbreak lasted for about 7 days, and was as severe or evenslightly more severe than a typical outbreak before the DNCB treatmentswere undertaken. The outbreak seemed to worsen as the inflammationassociated with the DTH response worsened, and only lessened when theDTH response subsided.

Following that second DNCB treatment at the site of a lesion, thesubject had no outbreaks at all for 6 months, which he reports as thelongest he has ever gone without a cold sore. At the end of this 6-monthperiod, he had a mild cold sore and chose to apply a 0.1% DNCB solutionto the cold sore. Again, this induced a moderate DTH response, and theinflammation seemed to worsen the outbreak, so the outbreak lasted about10 days. When the outbreak subsided, the subject went another 6 monthswithout any outbreaks to the present time.

For several months before and for the entire time described herein afterthe initial DNCB treatment, the subject took no antiviral medicines.

Example 2 Treatment of Herpes Labialis by Squaric Acid Dibutyl Ester

Thirty patients are recruited meeting the following criteria:

Inclusion Criteria Exclusion Criteria 18-85 years old Men andnon-pregnant/ Pregnancy or attempting to become non-lactating womenpregnant Able to understand and comply Unable to return for follow-upvisits or with all requirements of protocol comply with protocol Six ormore episodes of herpes Prior treatment with Squaric Acid and labialisof a recurring nature current active therapy per year Cancer treatmentand any immunodeficiency

In a screening interview, vital signs are recorded, and information iscollected on any medications the patient is currently taking, previousand current treatments for cold sores, severity and duration of coldsores.

Visit 1:

Upon entering the study, patients are sensitized to squaric acid dibutylto ester (SADBE) by dipping a cotton-tipped swab in a 2% solution ofSADBE in DMSO and swabbing a 1 cm² area on the forearm. Participants aretold then to wait at least 2 weeks for their first treatment.

Throughout the study, the participants are asked to maintain a subjectdiary, in which they record each day any symptoms they experience,including fever, swelling, pain, redness, itching, burning, the size ofany lesions, and any medications taken.

Visit 2:

After the 2-week period to allow development of sensitivity to SADBE,upon the beginning of his or her next outbreak (within 72 hours of thefirst signs of an outbreak), each participant is swabbed at the site ofthe herpes labialis lesion with a 0.5% solution of SADBE in DMSO.

Visit 3:

Upon the next distinct outbreak at least 2 weeks after the previoustreatment at visit 2, study diaries are collected. If the diary reflectsredness, blistering, or burning greater than 0.5 cm beyond the clinicallesion with the previous SADBE treatment, the dosage of SADBE isdecreased to 0.1%. If the diary reflects no redness or inflammationattributable to SADBE treatment, dosage is increased to 2%. Otherwise,dosage is maintained at 0.5%. Participants are swabbed at the site ofthe herpes labialis lesion with the SADBE solution.

If the hypersensitivity reaction produced by SADBE is excessive,patients are treated with a topical steroidal antiinflammatory cream.When a patient requests, outbreaks are treated with oral valacycloviruntil the outbreak resolves.

For at least 6 months after visit 2, patients maintain their diaries.Every two months the diaries are collected.

At visit 1, before applying SADBE, a blood sample is collected from eachpatient for use in a peripheral blood mononuclear cell (PBMC)proliferation assay to test immune response to herpes virus.

Two months after visit 2 (the first treatment application of SADBE to anHSV lesion) if the patient has not experienced a recurrence outbreakafter visit 2, or one month after visit 3 (the treatment application ofSADBE to a lesion of a second outbreak), a blood sample is collectedfrom the patient. The first, pretreatment, blood sample and the second,posttreatment, blood sample are used to conduct PBMC proliferationassays to test immune response to herpes virus before and aftertreatment.

PBMC Proliferation Assay

Venous blood is collected in heparinized test tubes for mononuclear cellisolation prior to treatment and after treatment at the times describedabove. Sample specimens are immediately transferred to the laboratoryfor processing.

Venous blood (15 ml) is transferred to a 50 ml centrifuge tube, dilutedto a total volume of 30 ml with saline or PBS, underlayed withFico/Lite-LymphoH™ (Atlanta Biologicals) and centrifuged for 20 minutesat 2100 rpm in an Eppendorf Model 5804R centrifuge. Interface cells arecollected and washed 2× with saline/PBS, centrifuged and resuspended insaline/PBS. Cell counts are performed using a Beckman Coulter Z1particle counter and the cells resuspended in freezing media (RPMI/20%human AB serum) and stored at −70° C. Pre- and post-treatment peripheralblood mononuclear cells from each patient are stored for proliferationassays.

KOS HSV-1 virus (American Type Culture Collection) is grown in culturein VERO cells and collected. Virus is filtered through a 45:m filterattached to a 3 ml syringe, into sterile cryovials. The titer of thevirus stock is determined. Virus is then heat inactivated, and stored at−20° C. for use to stimulate PMBC in the proliferation assays.

Peripheral blood mononuclear cells are thawed, washed 2× in saline/PBSand resuspended at 5×10⁵ cell/ml in RPMI/10% human AB serum. Cells areplated at 200 l/well. Heat inactivated KOS HSV-1 particles (2×10⁵ pfuper well) are added to the experimental wells. Concanavalin A (5:g/ml)is added to other wells as a positive control. Negative control wellshave no additions. Plates are incubated at 37° C., 5% CO₂ for 5 days andthen assayed for proliferation with Cell Counting Kit 8, a tetrazoliumdye assay (Dojindo Molecular Technologies, Gaithersburg, Md.).

Results are calculated by averaging optical density of the wells in eachof the groups. A stimulation index is calculated to reflect theproliferation in the wells stimulated by killed HSV as compared to thepositive and negative controls. The average optical absorbance of thepositive controls is set as a stimulation index (SI) value of 100 andthe average absorbance of the negative controls as a SI value of 0.

Results:

A total of 46 patients have been enrolled in the study and have notdropped out.

The groups are:

Group name Sensitization solution Treatment solution A (Placebo) 0%SADBE in DMSO   0% SADBE in DMSO B 2% SADBE in DMSO 0.5% SADBE in DMSO C2% SADBE in DMSO 0.2% SADBE in DMSOSurprisingly, it was found that persons in the treatment groups B and Creceiving 2% SADBE in DMSO as a sensitization dose usually did not havea subsequent outbreak during the study time, whereas those in theplacebo group A usually did. This is shown in Table 1.

TABLE 1 Proportion of subjects in each treatment group having a coldsore outbreak within 120 days after receiving the sensitization dose.Persons having an outbreak within X days after the sensitization dose pvalue for A Group proportion A versus B + C Days (Placebo) B C B + Cproportion 30 days 5 (of 15) 2 (of 16) 3 (of 15) 5 0.26 (of 31) 45 days8 (of 15) 4 (of 16) 4 (of 15) 8 0.099 (of 31) 60 days 9 (of 15) 5 (of16) 4 (of 15) 9 0.0583 (of 31) <120 days  11 6 (of 16) 4 (of 15) 100.0125* (of 15) (of 31) Total 15 16 15 31 persons in group*statistically significant (p < 0.05)The p value was calculated using the Fischer's two-tailed exact test.

In addition, the proportion in group A who had a first outbreak between30 and 120 days after receiving the sensitization dose was 4 of 14,while the proportion in pooled groups B and C was 5 of 26, and thisdifference is also statistically significant. (p<0.05).

The average days to the first outbreak after the sensitization dose wasalso measured. If a patient had not had an outbreak by 120 days, a valueof 120 days was used. The A: placebo, 0% SADBE in DMSO.

TABLE 2 Average days to first outbreak after the sensitization dose(using 120 days if a subject has not had an outbreak in 120 days.) Daysto 1st outbreak Group A (Placebo) B C B + C Average 63.0* 88.5 94.791.5* Standard 41.80 40.86 48.4 44.02 deviation n 15 16 15 31 <120 days11 (of 15) 6 (of 16) 4 (of 15) 10 (of 31) Total in 15 16 15 31 study*statistically significant difference, p < 0.05, t test.

The difference in average days between group A (placebo) and the pooledgroups B and C is statistically significant (p=0.041).

Table 3 shows the days to the first new outbreak from the date of thesensitization dose (2% SADBE or placebo applied to the inner aspect ofthe upper arm) in each patient.

TABLE 3 Days to first new outbreak after sensitization dose on upper armPlacebo group 2% SADBE 19 14 20 14 24 15 25 18 28 34 40 37 40 41 42 5056 80 64 91 98 >120107  >120 >120  >120 >120  >120 >120  >120 >120 >120 >120 >120 >120 >120 >120 >120 >120 >120 >120 >120 >120 >120median = 42 median = days greater than 120 days

The median time to the next outbreak in the placebo group was 42 days,while in the treatment group it was greater than 120 days. If theoutbreaks occurring in the 21 days after the sensitization dose areexcluded, the median time to the next outbreak was 56 days in theplacebo group and again over 120 days in the treatment group, countingfrom the day of the sensitization dose. Counting from day 21 after thesensitization dose, the median time to the next outbreak was 35 days (56minus 21) for the placebo group and greater than 99 days for thetreatment group.

Thus, a single sensitization dose of 2% SADBE in DMSO on the upper armsignificantly reduced time to outbreaks and the proportion of personswho had an outbreak at all for at least 120 days. It prevented futureoutbreaks. This occurred even without a treatment dose applied to thelip during an outbreak.

Many of the subjects had a rash (erythema) from the sensitization dose,which was not expected. In the placebo group A, 2 of 13 had a rash, andin the pooled treatment groups B and C, 17 of 28 had a rash (not allsubjects reported whether they had a rash or not, which is why the totalnumber of subjects here differs from Tables 1 and 2).

All patents, patent applications, and other publications cited areincorporated by reference.

What is claimed is:
 1. A method of treating herpes simple virus (HSV)infection comprising: an administering step comprising: (a) applying atopical immunosensitizer to the skin of a person infected with HSV at atime when the person is not having an HSV outbreak, or applying atopical immunosensitizer to the skin of a person infected with HSVduring an HSV outbreak wherein the applying is at a site other than asite of an HSV lesion; or (b) intradermally injecting a recall antigeninto a person infected with HSV at a time when the person is not havingan HSV outbreak, or intradermally injecting a recall antigen into aperson infected with HSV at a site other than a site of an HSV lesionduring an HSV outbreak; wherein the method does not further comprise (i)applying a topical immunosensitizer to the skin of a person infectedwith HSV on an HSV lesion at a time of an HSV outbreak in the person; or(ii) intradermally injecting a recall antigen into a person infectedwith HSV in an HSV lesion at the time of an HSV outbreak in the person;wherein the method reduces the frequency, duration, or severity of HSVoutbreaks in the person after the administering step.
 2. The method ofclaim 1 wherein the method reduces the frequency of new HSV outbreaks inthe person after the administering step.
 3. The method of claim 1wherein the method comprises applying a topical immunosensitizer to theskin of a person infected with HSV at a time when the person is nothaving an HSV outbreak, or applying a topical immunosensitizer to theskin of a person infected with HSV during an HSV outbreak wherein theapplying is at a site other than a site of an HSV lesion.
 4. The methodof claim 1 wherein the administering step comprises applying the topicalimmunosensitizer to skin on the inner aspect of the upper arm of theperson or intradermally injecting a recall antigen into skin on theinner aspect of the upper arm of the person.
 5. The method of claim 4wherein the administering step comprises applying the topicalimmunosensitizer to skin on the inner aspect of the upper arm of theperson.
 6. The method of claim 3 wherein the topical immunosensitizercomprises squaric acid, a squaric acid ester, diphenylcyclopropenone(DPCP), 1-chloro-2,4-dinitrobenzene (DNCB), or1-chloro-2,6-dinitrobenzene.
 7. The method of claim 3 wherein thetopical immunosensitizer comprises urushiol.
 8. The method of claim 6wherein the topical immunosensitizer comprises squaric acid dibutylester (SADBE).
 9. The method of claim 6 wherein the topicalimmunosensitizer comprises SADBE or a squaric acid diester.
 10. Themethod of claim 9 wherein the topical immunosensitizer is SADBE and isapplied as a solution in DMSO.
 11. The method of claim 1 wherein the HSVis HSV-1.
 12. The method of claim 1 wherein the HSV is HSV-2.
 13. Themethod of claim 1 or claim 2 wherein the person has herpes labialis. 14.The method of claim 1 wherein the person has genital herpes.
 15. Themethod of claim 1 wherein the method comprises: (b) intradermallyinjecting a recall antigen into a person infected with HSV at a timewhen the person is not having an HSV outbreak, or intradermallyinjecting a recall antigen into a person infected with HSV at a siteother than a site of an HSV lesion during an HSV outbreak; and therecall antigen comprises mumps antigen, candida antigen, or trichophytonantigen.
 16. The method of claim 1 wherein the method decreases time tohealing of lesions in a subsequent outbreak of the HSV infection afterstep (a) by at least 30%.
 17. The method of claim 1 wherein the methodincreases time between outbreaks by at least 2-fold.
 18. The method ofclaim 17 wherein the method increases time between outbreaks by at least4-fold.
 19. The method of claim 1 wherein the method at least doublesstimulation index of peripheral blood mononuclear cells (PBMC) from apatient in a proliferation test with stimulation of proliferation bykilled HSV particles as compared to the stimulation index of PBMC fromthe patient before treatment.
 20. The method of claim 1 wherein themethod increases median time to the next outbreak after theadministering step by at least 2-fold.
 21. The method of claim 1 whereinthe method increases median time from day 21 following the administeringstep to the next outbreak that begins at least 21 days after theadministering step by at least 2-fold.
 22. The method of claim 1 whereinthe method increases median time from day 21 following the administeringstep to the next outbreak that begins at least 21 days after theadministering step by at least 4-fold.
 23. The method of claim 1 whereinthe method further comprises a second administering step comprising: (a)applying a topical immunosensitizer to the skin of a person infectedwith HSV at a time when the person is not having an HSV outbreak, orapplying a topical immunosensitizer to the skin of a person infectedwith HSV during an HSV outbreak wherein the applying is at a site otherthan a site of an HSV lesion; or (b) intradermally injecting a recallantigen into a person infected with HSV at a time when the person is nothaving an HSV outbreak, or intradermally injecting a recall antigen intoa person infected with HSV at a site other than a site of an HSV lesionduring an HSV outbreak.
 24. The method of claim 23 wherein the secondadministering step occurs 7 days to 30 days after the firstadministering step.
 25. The method of claim 23 wherein the secondadministering step occurs 7 days to 365 days after the firstadministering step and occurs at a time when the person is having an HSVoutbreak.
 26. The method of claim 23 wherein the first and secondadministering steps both comprise: (a) applying a topicalimmunosensitizer to the skin of a person infected with HSV at a timewhen the person is not having an HSV outbreak, or applying a topicalimmunosensitizer to the skin of a person infected with HSV during an HSVoutbreak wherein the applying is at a site other than a site of an HSVlesion.
 27. The method of claim 26 wherein the topical immunosensitizercomprises SADBE or a squaric acid diester.
 28. The method of claim 1wherein the administering step produces an erythema in the person at thesite of administering.
 29. The method of claim 1 wherein theadministering step does not produce an erythema in the person at thesite of administration.
 30. The method of claim 1 wherein theadministering step comprises applying the topical immunosensitizer toskin on the spine of the person or skin over the trigeminal ganglion ofthe person or intradermally injecting a recall antigen into skin on thespine of the person or skin over the trigeminal ganglion of the person.31. A method of treating herpes simple virus (HSV) infection comprising:an administering step comprising: (a) applying a topicalimmunosensitizer to the skin of a person infected with HSV at a siteother than the genitals or lips, or to the genitals or the lips but at atime when the person is not having an HSV outbreak on the genitals orlips; or (b) intradermally injecting a recall antigen into a personinfected with HSV at a site other than the genitals or lips, or into thegenitals or the lips but at a time when the person is not having anoutbreak on the genitals or lips; wherein the method does not furthercomprise (i) applying a topical immunosensitizer to the skin of a personinfected with HSV on an HSV lesion at a time of an HSV outbreak in theperson; or (ii) intradermally injecting a recall antigen into a personinfected with HSV in an HSV lesion at the time of an HSV outbreak in theperson; wherein the method reduces the frequency, duration, or severityof HSV outbreaks in the person after the treatment step.
 32. The methodof claim 31 wherein the method reduces the frequency of new HSVoutbreaks in the person after the administering step.
 33. A method oftreating herpes simple virus (HSV) infection comprising: anadministering step comprising: (a) applying a topical immunosensitizerto the skin on the inner aspect of the upper arm of a person infectedwith HSV; or (b) intradermally injecting a recall antigen into the skinon the inner aspect of the upper arm of a person infected with HVS;wherein the method reduces the frequency, severity, or duration of HSVoutbreaks in the person after the administering step.
 34. The method ofclaim 33 wherein the method reduces the frequency of new HSV outbreaksin the person after the administering step.
 35. The method of claim 33wherein the administering step comprises: (a) applying a topicalimmunosensitizer to the skin on the inner aspect of the upper arm of aperson infected with HSV.
 36. The method of claim 33 wherein the methoddoes not further comprise (i) applying a topical immunosensitizer to theskin of a person infected with HSV on an HSV lesion at a time of an HSVoutbreak in the person; or (ii) intradermally injecting a recall antigeninto a person infected with HSV in an HSV lesion at the time of an HSVoutbreak in the person.
 37. A medical use of a substance capable ofinducing a delayed-type hypersensitivity (DTH) response in humans toprepare a medicament effective to reduce the frequency of subsequentherpes simplex virus (HSV) outbreaks in a patient with HSV infectionwhen administered to the skin of a person infected with HSV at a timewhen the person is not having an HSV outbreak or administered to theskin of a person infected with HSV during an HSV outbreak wherein theadministering is at a site other than a site of an HSV lesion.
 38. Themedical use of claim 37 wherein the substance is a topicalimmunosensitizer capable of inducing a DTH response in humans whenadministered topically.
 39. The medical use of claim 38 wherein thetopical contact sensitizer is squaric acid dibutyl ester,diphenylcyclopropenone (DPCP), 1-chloro-2,4-dinitrobenzene (DNCB), or1-chloro-2,6-dinitrobenzene.
 40. The medical use of claim 38 wherein thetopical immunosensitizer is urushiol.
 41. The medical use of claim 38wherein the medicament is a solution of SADBE in DMSO.
 42. The medicaluse of claim 37 wherein the substance is a recall antigen.
 43. Themedical use of claim 42 wherein the recall antigen comprises mumpsantigen, candida antigen, or trichophyton antigen.
 44. A medical use ofa topical contact sensitizer or a recall antigen to prepare a medicamenteffective to reduce the frequency of subsequent herpes simplex virus(HSV) outbreaks in a patient with HSV infection when administered by (a)applying a topical immunosensitizer to the skin on the inner aspect ofthe upper arm of a person infected with HSV; or (b) intradermallyinjecting a recall antigen into the skin on the inner aspect of theupper arm of a person infected with HSV.
 45. A composition comprising atopical immunosensitizer or a recall antigen for use in a method oftreating HSV infection, the method comprising: an administering stepcomprising: (a) applying a topical immunosensitizer to the skin of aperson infected with HSV at a time when the person is not having an HSVoutbreak, or applying a topical immunosensitizer to the skin of a personinfected with HSV during an HSV outbreak wherein the applying is at asite other than a site of an HSV lesion; or (b) intradermally injectinga recall antigen into a person infected with HSV at a time when theperson is not having an HSV outbreak, or intradermally injecting arecall antigen into a person infected with HSV at a site other than asite of an HSV lesion during an HSV outbreak; wherein the method doesnot further comprise (i) applying a topical immunosensitizer to the skinof a person infected with HSV on an HSV lesion at a time of an HSVoutbreak in the person; or (ii) intradermally injecting a recall antigeninto a person infected with HSV in an HSV lesion at the time of an HSVoutbreak in the person; wherein the method reduces the frequency of newHSV outbreaks in the person after the administering step.
 46. Acomposition comprising a topical immunosensitizer or a recall antigenfor use in a method of treating HSV infection, the method comprising: anadministering step comprising: (a) applying a topical immunosensitizerto the skin on the inner aspect of the upper arm of a person infectedwith HSV; or (b) intradermally injecting a recall antigen into the skinon the inner aspect of the upper arm of a person infected with HSV;wherein the method reduces the frequency of new HSV outbreaks in theperson after the administering step.